The present invention relates to new applications of lysozyme dimer and to compositions containing such dimer. The new applications are based on a common principle of non-specific stimulation of the immune system and are particularly useful for the prevention and/or treatment of symptoms or diseases in connection with an impaired function of the natural defensive and regenerative systems in the human and animal body. In particular, the present invention relates to uses of the lysozyme dimer to restore a suppressed immune system in humans and animals.
In the late 1980s, it was discovered that the dimerized forms of certain enzymes, while substantially retaining the beneficial properties of the corresponding monomers, turned to be by far less toxic than the monomers themselves and in some instances did not even display negative side effects at all when used in therapeutic doses. Antiviral and antibacterial compositions comprising as the active ingredient lysozyme dimer or other dimerized enzymes have been described in WO 89/11294. It is reported therein that lysozyme dimer is capable of inhibiting proliferation of a number of bacterial strains cultivated on samples taken from patients, when applied in concentrations of 1.25-20 mg/ml of the culture. It is also reported that the dimer is effective in treating canine parvovirus (CPV) infections when administered orally twice a day at a dose of 1-2 mg/kg of body weight. Later on, further attractive features of lysozyme dimers were found and additional therapeutical applications of the drug were developed, especially for the treatment of bacterial and viral infections as disclosed, for instance, in WO 94/01127.
In WO 94/01127, a model theory is presented that can help to understand the different effects observed with the lysozyme dimer. Although the entire mode of action of the lysozyme dimer is not yet fully understood, it appears that there is additional curative capability that cannot be explained by the bacteriolytic activity of the corresponding monomer. The inventors observed certain immunostimulative effects of the dimerized lysozyme, particularly concerning the modulation of cytokine levels. Moreover, from their experiments, they concluded that lysozyme dimer seems to prevent the penetration of bacterial cells by viruses, presumably by blocking certain regions of the outer cell surface and probably comprising virus receptor proteins.
The prior art discloses further results obtained in vitro with lysozyme dimer. Particularly, Bartholeyns and Zenebergh (Europ. J. Cancer, Vol.15, 1979, 85-91) tested dimerized lysozyme for cytostatic activities against liver cancer cells (HCT) in vitro. They observed a 73%.+-.15% inhibition of cancer cell multiplication in the cell culture (ibid., p.89, Table 2).
Surprisingly, except for WO 94/01127, no in vivo experiments with lysozyme dimer are reported so far. It is very strange and astonishing, and up to now waits for explanation, why neither Bartholeyns and Zenebergh nor any other researcher resumed this subject to promote further development of a promising discovery to combat cancer. A comparative showing (FIG. 1) of the purity of lysozyme dimer produced according to the method of Sorrentino et al., Eur. J. Biochem. 124, 183-189 (1982) and of the lysozyme dimer preferably used in the present invention revealed at least one possible reason: high concentrations of by-products such as lysozyme monomer, trimer and tetramer are found in the preparation produced according to Sorrentino et al., whereas the product preferably used in the present invention is highly purified, i.e., contains the desired lysozyme dimer in amounts of up to 90% or more by weight of the total lysozyme fraction of the preparation. A process for the manufacture of such highly purified lysozyme dimer has been described in WO 91/10731, the entire disclosure of which is incorporated herein by reference. This strongly supports the assumption that the purity of the prior art lysozyme dimer was simply not good enough for in vivo experiments and applications because it was known in the art already over 15 years ago that the monomeric form of lysozyme, despite its beneficial antibacterial activity, is rather toxic and can cause inflammations and severe allergies and even toxic shock symptoms.
In light of such circumstances it appears more understandable why no competent researcher including Bartholeyns and Zenebergh--although recommending lysozyme dimer as a promising candidate for further investigations--has carried out further experiments during the past ten to fifteen years to develop lysozyme dimer applications in vivo.
In spite of such lack of research activities of the scientific world possibly due to a prejudice of the art against the use of lysozyme dimer in vivo, the present inventors carried out further research and developmental work to improve the method of production and purification of the dimerized lysozyme and to find in vivo human and animal applications for the product, which led, for instance, to the antiviral and antibacterial and TNF level modulating applications disclosed in WO 94/01127, and the entire disclosure of which is incorporated herein by reference.
Use of immunostimulants, adjuvants and vaccines offers a wide range of attractive methods for inducing and building up protection against diseases. In this respect, "immunostimulants" refer to compounds that only stimulate non-specific defense mechanisms and protect against diseases. "Immunomodulators" refer to compounds that regulate (or modulate) the defense mechanisms after suppression (or decrease of immunity) of those mechanisms. Such suppression can arise from or be induced by many sources including pollutants, chemotherapeutics, stress, food, temperature changes, and the like. Immunomodulators stabilize the defense mechanisms after the influence of pathogens, and increase cellular and humoral immunity. Some immunomodulators are also able to depress and/or normalize hyperactive defense mechanisms including modulation of cytokine levels. Many immunostimulants are also classified as immunomodulators.
In the field of vaccine medicine, it is common that the patient exhibits only a small positive reaction to the vaccine, which thus provides only minimal protection. Many studies have shown or theorized that the effect of a vaccine is determined by the level of cell-mediated immunity and activity of cells for the production of specific antibodies. It has been known that many factors, such as products, drugs, particularly antibiotics, pollutants and stress, decrease the level of defense mechanisms and cell-mediated immunity. Thus if the humoral response mechanisms are suppressed, effectiveness of a subsequently administered vaccine can be reduced or even eliminated.
In an effort to overcome these problems, adjuvants have typically been used in combination with vaccine preparations. Adjuvants are substances used to enhance the specific immune response. Adjuvants are generally mixed and injected with antigen preparations, acting to elevate the specific immune activity. That is, the adjuvants generally are used to increase the level of antibody secreting cells circulating antibody titres. Adjuvants thus generally potentiate the specific immune response and stimulate production of specific antibodies.
Complete Freund's adjuvant is the classic adjuvant, inducing an inflammatory response at the injection site. Freund's adjuvants, combining paraffin oil and killed tubercle bacilli, were among the first immunostimulants used in humans and animals that were mixed with immunogens. Lanolin, paraffin or other various light oils were also used in conjunction with bacterin or vaccines. Unfortunately, these oil adjuvants can not be used as immunostimulants in routine immunization because, by their very nature, adverse inflammatory reactions often occur at the injection site. One example of a suitable adjuvant, approved by the U.S. Food and Drug Administration for use in humans in the United States, is alum or aluminum-phosphate precipitate, as used with diphtheria toxin.
Moreover, based on their knowledge of low toxicity of dimerized lysozyme compared to the monomer and on the availability of a new, highly purified lysozyme dimer preparation, the present inventors attempted and started anti-cancer trials with lysozyme dimer preparations in vivo, although the prior art did not suggest its use to treat diseases other than bacterial or viral infections. It is an advantage of the present invention to provide for the use of a highly purified lysozyme dimer for the manufacture of a pharmaceutical composition to supplement or replace the extremely toxic anti-cancer drugs usually applied in conventional chemotherapy.
Further investigative work by the present inventors has also revealed additional advantageous uses of the lysozyme dimer. Recent studies on immunomodulating properties of the lysozyme dimers have shown that it potentiates humoral response in mammals. In particular, the lysozyme dimer has been found to have the unexpected advantage of being capable of positively affecting the primary humoral response of mammals immunized with an antigen after immunosuppression. Such immunomodulating properties of the lysozyme dimer were not taught or suggested by the prior art.
Accordingly, in this respect, the present invention provides a method for increasing the cellular and humoral immunity response mechanisms after their suppression by some means such as toxic effects of antibiotics or chemicals. This is a completely new idea in the area of immunology. The concept of the present invention is to modulate non-specific cellular and humoral defense mechanisms, which form the first line of protection in immunology.
In particular, the present invention is directed to a method for treating or preventing a disease, comprising administering an effective amount of a pharmaceutical composition comprising a lysozyme dimer, wherein said lysozyme dimer contains about 10% by weight or less of unintended byproducts and is essentially free from a monomeric form of lysozyme, and wherein the disease is selected from the group consisting of cancer, hair growth disorder, a fish disease and a bee disease.
The present invention is also directed to a method for immunomodulating a humoral response in a human or animal subject to immunosuppression, comprising administering an effective amount of a pharmaceutical composition comprising a lysozyme dimer, wherein said lysozyme dimer contains about 10% by weight or less of unintended byproducts and is essentially free from a monomeric form of lysozyme.
These and other advantages are provided by the present invention.